Enzymatic characterization of D-lactate dehydrogenase and application in alanine aminotransferase activity assay kit

Figures & data

Figure 1. Enzyme catalysis reaction in ALT kit

Figure 2. Expression of ldLDH in E. coli and purification analysis

a) Schematic description of pET-28a-ldLDH expression plasmid. b) Expression of ldLDH at different temperatures and induction concentrations: the temperature was set at 16°C, 25°C, and 30°C; IPTG was used for expression induction at three different concentrations, 0.1 mM, 0.5 mM, and 1.0 mM. A strain cultured at 25°C without IPTG was used as control. After overnight incubation, the supernatant was used for SDS-PAGE analysis. c) Purification of ldLDH with a C-terminal His₆-tag by nickel affinity chromatography. D) SDS-PAGE analysis of purified ldLDH. lane 1, crude extract; lane 2, supernatant; lane 3, flow through; lane 4–5, purified ldLDH.

Figure 3. pH and temperature dependence of ldLDH activity

a) Effect of pH on the activity of ldLDH. Citrate buffer (pH 5.0–6.0), phosphate buffer (pH 6.0–7.5), Tris buffer (pH 7.5–9.0), carbonate buffer (pH 10.0–11.0). b) Effect of temperature on ldLDH activity. The activity of ldLDH was determined at the indicated temperatures. Data are expressed as a percentage of enzyme activity as assayed at 55°C. Error bars in the Figure indicate standard deviation from three parallel replicates.

Table 1. Effects of additives on ldLDH activity

No.	Additive	Conc.	Relative activity (%)
1	-	0	100.0 ± 0.6
2	NaCl2	1 mM	99.1 ± 1.6
3	KCl	1 mM	99.5 ± 1.8
4	CaCl2	1 mM	91.6 ± 2.4
5	MnCl2	1 mM	106.6 ± 5.1
6	FeCl3	1 mM	86.7 ± 4.4
7	MgCl2	1 mM	102.5 ± 3.1
8	ZnCl2	1 mM	96.8 ± 3.7
9	BaCl2	1 mM	82.5 ± 2.9
10	HgCl2	1 mM	43.8 ± 2.7
11	CuCl2	1 mM	63.2 ± 3.5
12	CoCl2	1 mM	97.5 ± 2.8
13	NiCl2	1 mM	96.9 ± 3.8
13	EDTA	1 mM	98.6 ± 5.2
14	Ethanol	1%	86.3 ± 3.3

Table 2. Comparison of biochemical properties of D-LDH from various strains

Strain	Specific activity (U/mg)	Optimum temperature (°C)	Optimum pH	Km (mM)	Kcat (s^−1)	Kcat/Km (mM^−1 s^−1)	Reference
Lactobacillus delbrueckii	1864	55	7.0–7.5	1.34	1603	1198	This study
Pediococcus acidilactici	442	30	5.5	0.09	287	3157	[7]
Pediococcus pentosaceus	835	45	5.5	0.49	320	658	[37]
Lactobacillus. coryniformis	1206	ND^a	ND	ND	ND	ND	[27]
sporolactobacillus inulinus	81.8	ND	5.5	ND	ND	ND	[36]
Lactobacillus. pentosus	18.6^b	ND	ND	0.12	321	2675	[10,11]
Lactobacillus. confusus	ND	45	6.0	0.68	ND	ND	[37]
Bacillus. coagulans	ND	ND	ND	2.2	23.6	11	[8]

^aND: not detected.

^bThe value was detected using crude enzyme.

Table 3. LdLDH stabilizer screening

No.	Stabilizer	Concentration	Relative activity after Lyophilization (%)	Relative activity after preserved at 37°C for 60 days (%)
1	-	0	100.0 ± 0.5	47.3 ± 2.9
2	Sucrose	5%	100.7 ± 3.2	99.5 ± 3.4
3	Trehalose	5%	100.2 ± 2.7	85.3 ± 4.1
4	Glucose	5%	99.4 ± 4.6	16.6 ± 1.1
5	Lactose	5%	94.3 ± 3.8	21.8 ± 0.7
6	Potassium gluconate	5%	101.6 ± 5.0	81.9 ± 4.6
7	Sorbose	150 mM	87.1 ± 3.6	5.7 ± 0.2
8	Sorbitol	200 mM	116.2 ± 6.2	88.9 ± 3.7
9	Xylitol	200 mM	121.0 ± 6.6	87.6 ± 5.1
10	Glycine	100 mM	84.1 ± 3.9	82.7 ± 4.8
11	Lysine	100 mM	84.7 ± 3.1	3.8 ± 0.1
12	Asparagine	100 mM	96.0 ± 4.2	99.1 ± 3.7
13	Methionine	100 mM	87.8 ± 3.2	77.8 ± 4.2
14	Threonine	100 mM	91.8 ± 5.1	74.4 ± 3.9
15	EDTA	1 mM	109.0 ± 6.3	84.2 ± 4.6
16	NaCl	50 mM	94.2 ± 5.6	79.2 ± 3.2
17	KCl	50 mM	102.1 ± 4.9	76.0 ± 3.9
18	MgCl2	50 mM	83.1 ± 3.9	14.3 ± 1.2
19	CaCl2	50 mM	80.6 ± 5.2	88.2 ± 5.7
20	BSA	1%	108.6 ± 6.8	70.3 ± 5.1
21	PEG-2000	5%	80.7 ± 4.7	64.9 ± 3.9
22	Glutamate	100 mM	111.2 ± 6.3	79.7 ± 4.1
23	Citric acid	200 mM	101.8 ± 5.2	87.3 ± 3.5

Figure 4. Lineweaver-Burk plot of ldLDH

Figure 5. Effects of pH and temperature on ldLDH stability

a) Stability of ldLDH was determined by incubating for 30 min at the indicated pH value and then assayed at pH 8.0. Data are expressed as a percentage of maximal enzyme activity. b) Effect of temperature on the stability of ldLDH. Error bars in the figure indicate standard deviation of three parallel replicates.

Figure 6. Performance of ldLDH in ALT kit

Serum ALT activity from 3–1,400 U/L (a) and 3 − 150 U/L (b) was measured using the ALT kit with pure ldLDH (ALT-ldLDH) and commercial ALT kit (ALT-MR). C) The relative deviation between ALT-ldLDH and ALT-MR was determined using a commercial kit (ALT-MR) as a standard control.

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